Abstract

Multiparameter
immunofluorescent labeling is the method of choice for sorting of cell
populations out of heterogeneous mixtures. However, downstream applications of
isolated cells are usually limited since fluorescence channels and epitopes are
blocked by the antibody fluorochrome conjugates utilized for the flow sorting
experiment. Accordingly, antibody fluorochrome conjugates
that would allow for a release of cell epitopes and a reuse of fluorescence
detection channels after cell sorting might be of high interest for certain
downstream applications. Herein, we describe for the first time new types of antibody
fluorochrome conjugates that enable a highly specific multiparameter cell
staining. Unlike conventional fluorochrome conjugated antibodies, the
introduced conjugates rely on recombinantly engineered antibody fragments and a
conjugation chemistry that facilitates the
complete release of the conjugates from the cell surface after the flow sorting
step. The resulting cells are free of labeling providing maximal flexibility in
downstream applications. We demonstrate this flexibility in the context of a
workflow for the isolation of highly pure human regulatory T cells (Tregs).
These cells were labeled with, for example, CD4-PE-Vio® 770, CD127-FITC and
CD25-APC conjugates to be enriched by flow sorting in high purity and cell yields.
Final detection of the target cells was achieved by intranuclear labeling with
Anti-FoxP3-Vio 667 since a selective removal of the CD25-APC conjugate from the
cell surface facilitated the reuse of the APC detection channel and enabled
maximum sensitivity for the identification of the targeted Treg cells after
flow sorting.